The long term goal of our work is to develop a vaccine strategy for influenza that is more effective against antigenic drift variants than the current updating of vaccine viruses, which is typically a year behind the circulating viruses. The overall goal of the work proposed for this funding period is to characterize the antigenic sites recognized by antibodies in human sera that are induced or recalled by vaccination or infection and to determine how antigenic variants are selected by polyclonal antibodies in the human population. The Specific Aims for the next granting period are based on a hypothesis that epitopes are not equally immunogenic and the bulk of the antibody response may be to a small subset of antigenic sites. Accumulation of mutations during antigenic drift may cause particular antigenic sites to become less or more immunodominant. Specific Aim 1 is to obtain and characterize panels of monoclonal antibodies (mAbs) that react with HA and NA of influenza H3N2 isolates from 2002 through the 5 year grant. Human mAbs will be used with panels of mouse mAbs generated to fill in any gaps. Specific Aim 2 is to map the epitopes on the HA and NA. Neutralizing epitopes are conformational and not mimicked by peptides. Conformational epitopes will be mapped by selection and characterization of escape mutants, competition assays, directed mutagenesis and X-ray crystallography of antigen-antibody complexes in a few cases. Specific Aim 3 is to determine the relative immunodominance in vivo of individual epitopes using mAbs from Aim 1 in competition assays with human sera to determine if certain antigenic sites are dominant in people after vaccination or infection, and if there are differences in elderly or immunosuppressed subjects. Specific Aim 4 is to determine if immunodominance changes as the virus undergoes antigenic drift over the 5 years of the grant. Antibodies in human sera against individual antigenic sites of drifted viruses will be measured from subjects vaccinated each year. Specific Aim 5 is to determine if immunogenicity can be altered, by making mutant viruses or antigens that are designed to suppress immunogenicity of particular epitopes. Will response to other epitopes then be enhanced? The results of these experiments will show if there is immunodominance of certain antigenic sites, if the immunodominance changes during antigenic drift, and if immunodominance can be systematically altered. This knowledge could ultimately be used to manipulate vaccine antigens so that antibodies would be made against a wider spectrum of neutralizing epitopes and be more effective against antigenic drift. PUBLIC HEALTH RELEVANCE: Influenza vaccines are safe but have varying effectiveness due to antigenic variation of circulating influenza viruses in the human population. Influenza vaccine is updated every year with new viruses but due to the time required to determine that a new strain is spreading and to adapt it for vaccine use, the vaccine is usually a year behind the virus. The goal of this research is to determine the mechanisms by which new influenza variants are selected in the human population and to use this knowledge to modify the vaccine so that it is effective against a broader range of variant viruses. The knowledge gained will be applicable to a new subtype of influenza if and when a new pandemic begins.